Label-free measurement of the yeast short chain TAG lipase activity by ESI-MS after one-step esterification

Triacylglycerol (TAG) lipases hydrolyze ester bonds in TAG and release diacylglycerol (DAG), monoacylglycerol (MAG), and FA. We present a one-step chemical derivatization method for label-free quantification of a mixture of TAG, DAG, and MAG following lipase assay by ESI-MS. Because the ionization efficiencies of TAG, DAG, and MAG are not identical, lipase reaction products, DAG and MAG, are derivatized to TAG species by esterifying their hydroxyl groups using acyl chloride, whose acyl chain contains one less (or one more) –CH2 group than that of substrate TAG. This resulted in three TAG species that were separated by 14 Da from one another and exhibited similar ion responses representing their molar amounts in the mass spectra. A good linear correlation was observed between peak intensity ratios and molar ratios in calibration curve. This method enables simultaneous quantification of TAG, DAG, and MAG in lipase assay and, in turn, allows stoichiometric determination of the concentrations of FAs released from TAG and DAG separately. By applying this strategy to measure both TAG and DAG lipolytic activities of the yeast Tgl2 lipase, we demonstrated its usefulness in studying enzymatic catalysis, as lipase enzymes often show dissimilar activities toward these lipids. Triacylglycerol (TAG) lipases hydrolyze ester bonds in TAG and release diacylglycerol (DAG), monoacylglycerol (MAG), and FA. We present a one-step chemical derivatization method for label-free quantification of a mixture of TAG, DAG, and MAG following lipase assay by ESI-MS. Because the ionization efficiencies of TAG, DAG, and MAG are not identical, lipase reaction products, DAG and MAG, are derivatized to TAG species by esterifying their hydroxyl groups using acyl chloride, whose acyl chain contains one less (or one more) –CH2 group than that of substrate TAG. This resulted in three TAG species that were separated by 14 Da from one another and exhibited similar ion responses representing their molar amounts in the mass spectra. A good linear correlation was observed between peak intensity ratios and molar ratios in calibration curve. This method enables simultaneous quantification of TAG, DAG, and MAG in lipase assay and, in turn, allows stoichiometric determination of the concentrations of FAs released from TAG and DAG separately. By applying this strategy to measure both TAG and DAG lipolytic activities of the yeast Tgl2 lipase, we demonstrated its usefulness in studying enzymatic catalysis, as lipase enzymes often show dissimilar activities toward these lipids. Triacylglycerol (TAG), a nonpolar storage lipid, serves as the most efficient energy source in diverse cells, provides building blocks for cell membrane lipid biosynthesis, and releases second messenger for cell signaling upon degradation (1.Wenk M.R. Lipomics: new tools and applications.Cell. 2010; 143: 888-895Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Thus, TAG mobilization plays a key role in energy homeostasis and cell proliferation in the biological systems. Lipolytic enzymes, lipases, are involved in TAG catabolism and deregulation of these activities perturbs cell physiology, leading to metabolic disorders and pathological conditions in many organisms. Lipases carry hydrolyzing activities toward TAG and diacylglycerol (DAG), forming DAG plus FA and monoacylglycerol (MAG) plus FA, respectively. Their catalytic activities to TAG and DAG can be very different, though. Because lipase assay produces a mixture of TAG, DAG, MAG, and FA, simultaneous quantification of these lipids is the first step in enzymatic kinetics. Traditional methods for the determination of lipolytic activity include pH titrimetry and TLC analysis (2.Beisson F. Tiss A. Rivière C. Verger R. Methods for lipase detection and assay: a critical review.Eur. J. Lipid Sci. Technol. 2000; 102: 133-153Crossref Google Scholar, 3.Stoytcheva M. Montero G. Zlatev R. Leon J.A. Gochev V. Analytical methods for lipase activity determination: a review.Curr. Anal. Chem. 2012; 8: 400-407Crossref Scopus (63) Google Scholar). The TLC-based method requires radioactive- or fluorescence-labeled substrates/products for detection and monitors the disappearance of substrates and/or the appearance of products on TLC plate. Either autoradiographic or densitometric analysis can be carried out for quantification. The titration method is an indirect assay, measuring the amount of base added to neutralize the released FA. The latter method is restricted to a specific pH range and less sensitive than the former one. While lipase activities are determined by the amount of FAs released from substrates per minute under standard condition, the methods targeting FAs are unable to distinguish hydrolyzing activities toward TAG and DAG. More recently, MS linked to LC has been widely used in the field of lipid research (4.Ivanova P.T. Milne S.B. Myers D.S. Brown H.A. Lipidomics: a mass spectrometry based systems level analysis of cellular lipids.Curr. Opin. Chem. Biol. 2009; 13: 526-531Crossref PubMed Scopus (128) Google Scholar, 5.Blanksby S.J. Mitchell T.W. Advances in mass spectrometry for lipidomics.Annu. Rev. Anal. Chem. (Palo Alto Calif). 2010; 3: 433-465Crossref PubMed Scopus (253) Google Scholar, 6.Wang M. Wang C. Han R.H. Han X. Novel advances in shotgun lipidomics for biology and medicine.Prog. Lipid Res. 2016; 61: 83-108Crossref PubMed Scopus (179) Google Scholar). In particular, ESI-MS has greatly facilitated the quantitative and sensitive analysis of both polar and neutral lipids since the first reports of phospholipid analysis (7.Han X. Gross R.W. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.Proc. Natl. Acad. Sci. USA. 1994; 91: 10635-10639Crossref PubMed Scopus (378) Google Scholar, 8.Kim H.Y. Wang T.C. Ma Y.C. Liquid chromatography/mass spectrometry of phospholipids using electrospray ionization.Anal. Chem. 1994; 66: 3977-3982Crossref PubMed Scopus (223) Google Scholar). For instance, an LC-ESI-MS system has been applied to determine lipase activity by monitoring the FA production (9.Hao G. Yang L. Mazsaroff I. Lin M. Quantitative determination of lipase activity by liquid chromatography-mass spectrometry.J. Am. Soc. Mass Spectrom. 2007; 18: 1579-1581Crossref PubMed Scopus (10) Google Scholar). Further progress in lipid research has been made by combining chemical derivatization with high-resolution nano ESI-MS in lipidome analysis (10.Ryan E. Reid G.E. Chemical derivatization and ultrahigh resolution and accurate mass spectrometry strategies for "shotgun" lipidome analysis.Acc. Chem. Res. 2016; 49: 1596-1604Crossref PubMed Scopus (81) Google Scholar). TAG, DAG, and MAG are all uncharged fatty acyl ester derivatives of glycerol. In ESI-MS, this class of neutral lipids has been routinely analyzed as metal adduct ions after introducing charge states with alkali metals (11.Hsu F-F. Ma Z. Wohltmann M. Bohrer A. Nowatzke W. Ramanadham S. Turk J. Electrospray ionization/mass spectrometric analyses of human promonocytic U937 cell glycerolipids and evidence that differentiation is associated with membrane lipid composition changes that facilitate phospholipase A2 activation.J. Biol. Chem. 2000; 275: 16579-16589Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 12.Han X. Gross R.W. Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2001; 295: 88-100Crossref PubMed Scopus (295) Google Scholar). For analysis of DAG species, a derivatization method using N-chlorobetainyl chloride or N,N-dimethylglycine has significantly improved the quantification efficiency of ESI-MS (13.Li Y.L. Su X. Stahl P.D. Gross M.L. Quantification of diacylglycerol molecular species in biological samples by electrospray ionization mass spectrometry after one-step derivatization.Anal. Chem. 2007; 79: 1569-1574Crossref PubMed Scopus (57) Google Scholar, 14.Wang M. Hayakawa J. Yang K. Han X. Characterization and quantification of diacylglycerol species in biological extracts after one-step derivatization: a shortgun lipidomics approach.Anal. Chem. 2014; 86: 2146-2155Crossref PubMed Scopus (51) Google Scholar). By taking a MS-based shotgun lipidomics approach, the latter method allows accurate and reproducible analysis of all of the cellular DAG species, including 1,2- and 1,3-DAG isomers (14.Wang M. Hayakawa J. Yang K. Han X. Characterization and quantification of diacylglycerol species in biological extracts after one-step derivatization: a shortgun lipidomics approach.Anal. Chem. 2014; 86: 2146-2155Crossref PubMed Scopus (51) Google Scholar). However, TAG, DAG, and MAG carrying the same acyl side chain(s) show very dissimilar ion responses and, in turn, different peak intensities, making their simultaneous quantification difficult. Even within each species of a lipid class, detection sensitivity of ESI-MS varies significantly depending on the acyl chain length and the unsaturation index in addition to the experimental setups. Via simple esterification, here we report a strategy to obtain identical ion responses from TAG, DAG, and MAG that contain the same acyl side chain(s). We used ESI-MS to quantify a mixture of TAG, DAG, and MAG following chemical derivatization and investigated quantification linearity and detection sensitivity over a wide dynamic range. Biological application of this method was demonstrated by measuring lipolytic activities of the yeast Saccharomyces cerevisiae Tgl2 protein. Acyl chlorides (heptanoyl and nonanoyl chlorides), N-bromosuccinimide (NBS), 1-octanoyl-rac-glycerol (C8:0 MAG), 1,2-dioctanoyl-sn-glycerol (C8:0/C8:0 DAG), glyceryl trioctanoate (C8:0/C8:0/C8:0 TAG), and primuline were obtained from Sigma-Aldrich (St. Louis, MO). Acetic acid, acetone, anhydrous HPLC-grade dichloromethane (DCM), diethyl ether, hexane, and methanol were purchased from J. T. Baker (Center Valley, PA). BSA was from Merck (Darmstadt, Germany). Deionized distilled water was prepared by using a Mega-Pure system (Barnstead MP-6Am). The mixtures of C8:0/C8:0/C8:0 TAG, C8:0/C8:0 DAG, and C8:0 MAG were prepared at various molar ratios in a dry 4 ml vial by dissolving these lipids (113–900 nmol) with 900 μl of anhydrous DCM. Each mixture was subjected to esterification reaction by addition of 20 μl of acyl chloride (heptanoyl or nonanoyl chloride) and 20 μl of 5 mM NBS in DCM (15.Rezaei Z. Khabnadideh S. Zarshenas M.M. Jafari M.R. Esterification of tertiary alcohols in steroids under different conditions.J. Mol. Catal. A Chem. 2007; 276: 57-61Crossref Scopus (7) Google Scholar). The vial was flushed with dry nitrogen, capped, and incubated at 50°C for 7–12 h with stirring. The progress of esterification was monitored by lipid analysis on a TLC plate (Silica Gel 60 G plate; Merck) as described previously (16.Ham H.J. Rho H.J. Shin S.K. Yoon H-J. The TGL2 gene of Saccharomyces cerevisiae encodes an active acylglycerol lipase located in the mitochondria.J. Biol. Chem. 2010; 285: 3005-3013Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Lipid spots were identified under a UV lamp after spraying a primuline solution (5 mg in 100 ml of acetone/water; 80/20, v/v). When the reaction was complete, a sample solution was immediately diluted 20-fold with methanol containing 250 μM NaCl and analyzed by ESI-MS. In the preliminary experiments, we tested two esterification reagents (acid anhydride and acyl chloride) and two catalysts (NBS and pyridine) for the derivatization of DAG and MAG into TAG species and obtained optimum results with acyl chloride in the presence of NBS. Thus, we performed all the esterification reactions by using acyl chloride and NBS. We first determined reaction yield and rate constant for DAG/MAG esterification by ESI-MS. We prepared a 1:1:1 mixture of C8:0/C8:0/C8:0 TAG (0.9 μmol), C8:0/C8:0 DAG (0.9 μmol), and C8:0 MAG (0.9 μmol) in anhydrous DCM (900 μl) and performed esterification reactions by using heptanoyl chloride (129 μmol) or nonanoyl chloride (111 μmol) in the presence of NBS (0.1 μmol) at 50°C for 7 h. At times of 0, 1/6, 1/3, 2/3, 1, 2, 4, and 7 h, a small aliquot (50 μl) was taken from the reaction mixture and immediately diluted 20-fold with methanol (950 μl). The hydroxyl group of methanol was used to consume acyl chloride in excess and terminate the esterification reaction. The aliquots were subjected to ESI-MS to measure the relative abundances of TAG (C8:0/C8:0/C8:0 at m/z 494.2), TAG′ (C8:0/C8:0/C7:0 at m/z 480.1 or C8:0/C8:0/C9:0 at m/z 508.1), and TAG″ (C8:0/C7:0/C7:0 at m/z 466.1 or C8:0/C9:0/C9:0 at m/z 522.2). TAG′ and TAG″ were the outcomes of the DAG and MAG esterifications, respectively. We also took the ESI mass spectra of C8:0/C8:0/C8:0 TAG in the absence of C8:0/C8:0 DAG and C8:0 MAG after incubation with heptanoyl or nonanoyl chloride and confirmed that there were no peaks at m/z 480.1 and 466.1 or at m/z 508.1 and 522.2 (H. J. Ham et al., unpublished observations). Therefore, both TAG′ and TAG″ were not originated from the reagent C8:0/C8:0/C8:0 TAG. To purify hemagglutinin (HA)-tagged Tgl2 protein, yeast cells (strain YHY058d2; tgl2Δ) harboring the plasmid pYNO4-HA-TGL2 were cultured as described previously (16.Ham H.J. Rho H.J. Shin S.K. Yoon H-J. The TGL2 gene of Saccharomyces cerevisiae encodes an active acylglycerol lipase located in the mitochondria.J. Biol. Chem. 2010; 285: 3005-3013Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Yeast lysates (∼700 μl) prepared from ∼4 × 109 cells were incubated with 50 μl of anti-HA affinity matrix (clone 3F10, Roche) at 4°C overnight. The beads were washed extensively, resuspended with 200 μl of 20 mM sodium phosphate buffer (pH 7.4), and used for lipase assay. About 1/10 of HA-Tgl2 protein bound to the anti-HA affinity beads was analyzed by SDS-PAGE followed by SYPRO Ruby staining (Molecular Probes, Eugene, OR) and quantified using the fluorescence gel imaging system (VersaDoc 5000 MP; Bio-Rad). The amount of Tgl2 protein was determined from the calibration curve using BSA as an internal standard. Lipase assay was performed by the published method (16.Ham H.J. Rho H.J. Shin S.K. Yoon H-J. The TGL2 gene of Saccharomyces cerevisiae encodes an active acylglycerol lipase located in the mitochondria.J. Biol. Chem. 2010; 285: 3005-3013Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Briefly, C8:0/C8:0/C8:0 TAG was added to the assay buffer composed of 150 μl of 50 mM Tris-HCl (pH 8.0) and 20 μl of a BSA solution (20 mg/ml) to a final concentration of 1.74 mM (296 nmol). The mixture was sonicated at 37°C until the solution became cloudy (≥4 min) and kept at 37°C in the presence of 50 μl of 200 mM MgCl2 (total volume, 220 μl). The lipase reaction was initiated by adding 200 μl of HA-Tgl2 protein (4 μg) purified as above. Aliquots (20 μl each) were taken out at the start (time 0) and end (time 60 min) of incubation at 37°C and immediately analyzed by TLC to monitor the progress of the lipase reaction. Lipid species in the remaining reaction mixture (266 nmol; 380 μl of 0.7 mM) were extracted by adding 1 ml of chloroform and dried under N2 flow for chemical esterification. The residues were resuspended in DCM, esterified, and prepared for ESI-MS analysis as described above. The ESI mass spectra were obtained by using an ESI triple quadrupole mass spectrometer (AB SCIEX 2000 QTRAP; Forster City, CA). Sample solutions were directly transferred to the electrospray tip by using a syringe pump at a flow rate of 10 μl/min and electrosprayed with a N2 nebulizer gas. The electrospray voltage was 5 kV. Lipid-metal ion adducts were collected in the ion trap for 200 ms and scanned for 1.6 s to obtain a single scan spectrum in the m/z range 200–800. After 200 ms of dead time, ion collection was started again in the trap. A single scan spectrum was obtained every 2 s. The final ESI mass spectrum was constructed by summing total 30 single-scan spectra obtained for 1 min. All data were acquired in the positive ion mode. In ESI-MS, the ion response of lipid highly depends on the lipid structure and the mass range. TAG, DAG, and MAG containing the same acyl side chain(s) yield very different ion intensities due to their nonidentical ionization efficiencies. For instance, the ESI mass spectrum of a 1:1:1 mixture of TAG (C8:0/C8:0/C8:0), DAG (C8:0/C8:0), and MAG (C8:0) presents three major ions of [TAG + Na]+, [DAG + Na]+, and [MAG + Na]+ at m/z 494.2, 368.1, and 241.9, respectively (supplemental Fig. S1). The lipids were dissolved with NaCl in methanol to promote the formation of sodium ion adducts. As shown in supplemental Fig. S1, the peak intensities of [TAG + Na]+, [DAG + Na]+, and [MAG + Na]+ are not identical, although each lipid was mixed at equimolar concentration. Relative quantification of lipid species by ESI-MS requires similar ion responses representing their molar amounts. While the ionization efficiency is mainly determined by the lipid structure and the molecular weight, the same class of lipid species with one or two different carbon number(s) gives almost identical ion response. We thus esterified DAG and MAG to TAG species containing one and two less (or more) –CH2 group(s) than unmodified DAG and MAG, respectively, by using acyl chloride (supplemental Fig. S2). NBS was added to the reaction mixture to remove acidic byproduct and facilitate the esterification. The resulting TAG derived from DAG is named to TAG′ and the mass difference between TAG and TAG′ is 14 Da. Similarly, TAG″ is derived from MAG and the mass difference between TAG and TAG″ is 28 Da. When DAG or MAG whose carbon number in the acyl group is 8, for instance, is modified to TAG′ or TAG″ by using heptanoyl chloride (C7), whose carbon number is 7, a 2:1:2 mixture of TAG, TAG′, and TAG″ exhibits 2:1:2 peaks of sodium ion adducts at m/z 494.2, 480.1, and 466.1 in the mass spectra, respectively (supplemental Fig. S1C). Importantly, the peak intensity ratio faithfully reproduced the premixed molar ratio. Chemical esterification using nonanoyl chloride (C9), whose carbon number is 9, resulted in ion peaks of TAG, TAG′, and TAG″ at m/z 494.2, 508.1, and 522.2, respectively (supplemental Fig. S1D). These data suggest that our strategy minimizes the effects of lipid structure on ion response and enables simultaneous quantification of TAG, DAG, and MAG after lipase assay by using ESI-MS. Alternatively, the esterification can be performed by using stable isotope-labeled acyl chloride. Octanoyl chloride-d3 [CD3(CH2)6COCl], for instance, can convert DAG (C8:0/C8:0) and MAG (C8:0) to TAG-d3 (C8:0/C8:0/C8:0) and TAG-d6 (C8:0/C8:0/C8:0), respectively. Although this almost completely eliminates the structural effects on ion response, we prefer the use of label-free C7 or C9 because it is cost-effective and produces a mass difference of 14 Da (3 Da in the case of octanoyl chloride-d3) between TAG, TAG′, and TAG″. Ion peaks of these TAG species are well-separated in the mass spectra. The progress of the esterification reaction was checked by monitoring the relative ratios of TAG′ over TAG and TAG″ over TAG ([TAG′]/[TAG] and [TAG″]/[TAG]) in the ESI mass spectra as a function of reaction time (Fig. 1). When the lipid mixture was treated with C7, the measured ratios of [TAG′]/[TAG] and [TAG″]/[TAG] were 1.00 ± 0.19 and 0.97 ± 0.20 at 7 h, respectively (Fig. 1A). Thus, reaction yields for DAG and MAG esterifications were 100% and 97%, respectively. Both reactions were nearly completed at 2 h. In the case of incubation with C9, the measured ratios of [TAG′]/[TAG] and [TAG″]/[TAG] were 0.84 ± 0.11 and 0.70 ± 0.10 at 7 h, respectively, resulting in reaction yields of ∼84% and ∼70%, respectively (Fig. 1B). In this case, both DAG → TAG′ and MAG → TAG″ reactions plateaued after 4 h. The observed rate constant (kobs) for the appearance of TAG′ from DAG-to-TAG′ esterification was 3.83 h−1 using C7 (Fig. 1A) and 1.32 h−1 using C9 (Fig. 1B). Hence, the bimolecular rate constant for the reaction of DAG with acyl chloride was 8.25 cm3 mol−1 s−1 using C7 and 3.30 cm3 mol−1 s−1 using C9 after taking the concentration of acyl chloride into account in the equation {kobs/(3,600 s h−1)/[acyl chloride]}. In the case of consecutive MAG-to-TAG″ esterifications, the rate constant for the appearance of TAG″ from DAG′-to-TAG″ esterification can be assumed to be identical to that for the appearance of TAG′ from DAG-to-TAG′ esterification. Then the observed rate constant for the appearance of DAG′ from MAG-to-DAG′ esterification was 6.76 h−1 using C7 (Fig. 1A) and 1.80 h−1 using C9 (Fig. 1B), which resulted in the bimolecular rate constant of 14.56 cm3 mol−1 s−1 using C7 and 4.55 cm3 mol−1 s−1 using C9. Interestingly, the MAG-to-DAG′ esterification proceeded faster than the DAG-to-TAG′ esterification in both cases using C7 and C9. This is mainly because the hydroxyl group in DAG is more sterically hindered than those in MAG, indicating that the steric hindrance plays a significant role in the kinetics of esterification. It seems that chemical esterification using C7 resulted in higher yield and faster rate than that using C9 due to the shorter alkyl chain of C7. Our observation is in accord with the previous report on the esterification of tertiary alcohols in steroids using acyl chloride, exhibiting the reaction yield of 85–95% at the reaction time of 10–12 h (15.Rezaei Z. Khabnadideh S. Zarshenas M.M. Jafari M.R. Esterification of tertiary alcohols in steroids under different conditions.J. Mol. Catal. A Chem. 2007; 276: 57-61Crossref Scopus (7) Google Scholar). Both studies suggested that the reaction yield was higher and the reaction rate was faster, when the aliphatic chain length of acyl chloride was shorter. The kinetics of esterification can also be influenced by other parameters, such as catalyst, pH, solvent, temperature, and so on. We then examined the relationship between the measured ratios of TAG, TAG′, and TAG″ peak intensities and the predetermined molar ratios of TAG, DAG, and MAG to assess their simultaneous quantification after chemical esterification. The lipid samples (C8:0/C8:0/C8:0 TAG, C8:0/C8:0 DAG, and C8:0 MAG) were premixed in DCM at four different molar ratios (S1, 2:1:2; S2, 4:1:2; S3, 3:1:1; and S4, 8:1:2, respectively, of [TAG]:[DAG]:[MAG]) prior to derivatization. The sample mixtures S1–S4 were esterified using C7 or C9, and subsequently analyzed by ESI-MS. The relative amounts of TAG, DAG, and MAG were determined by measuring the peak heights of sodium ion adducts, [TAG + Na]+ at m/z 494.2, [TAG′ + Na]+ at m/z 480.1/508.1, and [TAG″ + Na]+ at m/z 466.1/522.2, respectively. We plotted the measured ratios of [TAG]/[TAG′] and [TAG]/[TAG″] against the predetermined ratios of [TAG]/[DAG] and [TAG]/[MAG], respectively (Fig. 2, Table 1). The data points obtained from S1–S4 were linearly fitted in the plots and the slopes were 0.98–1.03. These results clearly show a good linear correlation between the peak intensity ratios and the molar ratios for both DAG-to-TAG′ and MAG-to-TAG″ esterifications carried out by using C7 or C9. In line with this, the mole fractions expected from the premixed ratios of TAG, DAG, and MAG were faithfully reproduced by those of TAG, TAG′, and TAG″ observed in the mass spectra for both C7 and C9 incubations (Fig. 3, Table 2). The relationship between the observed and predetermined mole fractions was linear with the slopes of 1.00 (C7) and 1.03 (C9). In brief, a good linearity of relative quantification was obtained after chemical esterification, enabling simultaneous quantification of TAG, DAG, and MAG by ESI-MS.TABLE 1Summary of the molar ratios of TAG over DAG (TAG′ in the observed molar ratio) and TAG over MAG (TAG″ in the observed molar ratio)Predetermined Molar RatioObserved Molar RatioaErrors include an instrumental response error (5%).[TAG]/[DAG][TAG]/[MAG]C7C9[TAG]/[TAG′][TAG]/[TAG″][TAG]/[TAG′][TAG]/[TAG″]S1212.03 ± 0.101.03 ± 0.052.01 ± 0.110.94 ± 0.06S2423.86 ± 0.191.89 ± 0.094.23 ± 0.221.97 ± 0.12S3332.89 ± 0.143.10 ± 0.162.95 ± 0.163.09 ± 0.20S4847.97 ± 0.404.22 ± 0.218.61 ± 0.453.88 ± 0.25a Errors include an instrumental response error (5%). Open table in a new tab Fig. 3Quantification linearity in terms of mole fraction. Esterification using C7 (A); esterification using C9 (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Summary of the mole fractions of TAG, DAG (or TAG′), and MAG (or TAG″)Observed Mole FractionaErrors include an instrumental response error (5%).Predetermined Mole FractionC7C9TAGDAGMAGTAGTAG′TAG″TAGTAG′TAG″S10.400.200.400.41 ± 0.020.20 ± 0.010.39 ± 0.020.44 ± 0.020.21 ± 0.010.36 ± 0.02S20.570.140.290.55 ± 0.030.17 ± 0.010.29 ± 0.010.59 ± 0.030.19 ± 0.010.23 ± 0.01S30.600.200.200.60 ± 0.030.21 ± 0.010.19 ± 0.010.68 ± 0.030.15 ± 0.010.17 ± 0.01S40.730.090.180.73 ± 0.040.09 ± 0.010.17 ± 0.010.77 ± 0.040.08 ± 0.010.15 ± 0.01a Errors include an instrumental response error (5%). Open table in a new tab To investigate the sensitivity of quantification, we made a series of lipid mixtures by dilution of a master solution after esterification. Two master solutions of lipids (C8:0/C8:0/C8:0 TAG, C8:0/C8:0 DAG, and C8:0 MAG) were prepared at the molar ratios of 10:3:4 and 8:2:3 ([TAG]:[DAG]:[MAG]) and esterified using C7 and C9, respectively. Five-fold serial dilutions of each master solution were analyzed by ESI-MS (Tables 3, 4). Before or after dilution, the mole fraction ratios of TAG:TAG′:TAG″ are 0.59:0.18:0.24 and 0.62:0.15:0.23, as expected from the 10:3:4 and 8:2:3 premixed molar ratios of [TAG]:[DAG]:[MAG], respectively. After esterification using C7 (Table 3), the observed mole fractions determined by measuring signal intensities of TAG, TAG′, and TAG″ in the mass spectra were in good agreement with the expected ones (TAG:TAG′:TAG″ = 0.59:0.18:0.24) up to a mixture of 0.40 pmol of TAG, 0.12 pmol of TAG′, and 0.16 pmol of TAG″ and the signal-to-noise ratio was greater than 2. In the case of esterification using C9 (Table 4), the expected mole fraction ratio (TAG:TAG′:TAG″ = 0.62:0.15:0.23) was maintained up to a mixture of 16 fmol of TAG, 4 fmol of TAG′, and 6 fmol of TAG″ and the signal-to-noise ratio was greater than 3. Previously, a detection limit of 0.1 pmol was achieved for TAG by using ESI-MS (12.Han X. Gross R.W. Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2001; 295: 88-100Crossref PubMed Scopus (295) Google Scholar). Although the detection sensitivity is highly dependent on the instrumental conditions, our results obtained from the mixtures of TAG species by using an ESI triple quadrupole mass spectrometer are comparable to the results reported by others (12.Han X. Gross R.W. Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2001; 295: 88-100Crossref PubMed Scopus (295) Google Scholar). A further increase in the detection sensitivity could be achieved by adopting higher performance instruments.TABLE 3Summary of the expected concentrations of TAG, TAG′, and TAG″ and their mole fractions observed in the mass spectraExpected ConcentrationObserved Mole FractionaErrors include an instrumental response error (5%).[TAG][TAG′][TAG″]TAGTAG′TAG″150 μM (50 pmol)15 μM (15 pmol)20 μM (20 pmol)0.58 ± 0.010.18 ± 0.010.24 ± 0.01210 μM (10 pmol)3.0 μM (3.0 pmol)4.0 μM (4.0 pmol)0.59 ± 0.010.18 ± 0.020.23 ± 0.0232 μM (2.0 pmol)0.60 μM (0.60 pmol)0.80 μM (0.80 pmol)0.58 ± 0.030.18 ± 0.040.24 ± 0.0340.40 μM (0.40 pmol)0.12 μM (0.12 pmol)0.16 μM (0.16 pmol)0.58 ± 0.050.18 ± 0.060.24 ± 0.06580 nM (80 fmol)24 nM (24 fmol)32 nM (32 fmol)0.42 ± 0.110.22 ± 0.140.36 ± 0.18616 nM (16 fmol)4.8 nM (4.8 fmol)6.4 nM (6.4 fmol)0.38 ± 0.140.23 ± 0.110.39 ± 0.1973.2 nM (3.2 fmol)1.0 nM (1.0 fmol)1.3 nM (1.3 fmol)0.44 ± 0.100.21 ± 0.100.35 ± 0.11Five-fold serial dilutions were prepared from a 10:3:4 mixture of TAG, DAG, and MAG after esterification using C7. The mole fraction ratio of TAG:TAG′:TAG″ is expected to be 0.59:0.18:0.24.a Errors include an instrumental response error (5%). Open table in a new tab TABLE 4Summary of the expected concentrations of TAG, TAG′, and TAG″ diluted after C9 reaction and their observed mole fractionsExpected ConcentrationObserved Mole FractionaErrors include an instrumental response error (5%).[TAG][TAG′][TAG″]TAGTAG′TAG″150 μM (50 pmol)13 μM (13 pmol)19 μM (19 pmol)0.69 ± 0.030.14 ± 0.010.17 ± 0.05210 μM (10 pmol)2.5 μM (2.5 pmol)3.8 μM (3.8 pmol)0.66 ± 0.030.15 ± 0.020.19 ± 0.0432.0 μM (2.0 pmol)0.50 μM (0.50 pmol)0.75 μM (0.75 pmol)0.62 ± 0.030.16 ± 0.020.22 ± 0.0440.40 μM (0.40 pmol)0.10 μM (0.10 pmol)0.15 μM (0.15 pmol)0.61 ± 0.050.16 ± 0.040.24 ± 0.06580 nM (80 fmol)20 nM (20 fmol)30 nM (30 fmol)0.60 ± 0.090.16 ± 0.110.24 ± 0.10616 nM (16 fmol)4.0 nM (4.0 fmol)6.0 nM (6.0 fmol)0.63 ± 0.120.15 ± 0.070.22 ± 0.0873.2 nM (3.2fmol)0.80 nM (0.80 fmol)1.2 nM (1.2 fmol)0.40 ± 0.240.24 ± 0.190.36 ± 0.19A 8:2:3 mixture of TAG, DAG, and MAG was esterified. The mole fraction ratio of TAG: TAG′: TAG″ is expected to be 0.62:0.15:0.23.a Errors include an instrumental response error (5%). Open table in a new tab Five-fold serial dilutions were prepared from a 10:3:4 mixture of TAG, DAG, and MAG after esterification using C7. The mole fraction ratio of TAG:TAG′:TAG″ is expected to be 0.59:0.18:0.24. A 8:2:3 mixture of TAG, DAG, and MAG was esterified. The mole fraction ratio of TAG: TAG′: TAG″ is expected to be 0.62:0.15:0.23. We applied our esterification strategy to study lipolytic catalysis of the yeast Tgl2 protein. In the yeast Saccharomyces cerevisiae, TAG is mobilized by three lipases, Tgl3–5, present in the lipid droplets and the Tgl2 lipase located in the mitochondria (16.Ham H.J. Rho H.J. Shin S.K. Yoon H-J. The TGL2 gene of Saccharomyces cerevisiae encodes an active acylglycerol lipase located in the mitochondria.J. Biol. Chem. 2010; 285: 3005-3013Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 17.Athenstaedt K. Daum G. YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae.J. Biol. Chem. 2003; 278: 23317-23323Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 18.Athenstaedt K. Daum G. Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae are localized to lipid particles.J. Biol. Chem. 2005; 280: 37301-37309Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 19.Kurat C.F. Natter K. Petschnigg J. Wolinski H. Scheuringer K. Scholz H. Zimmermann R. Leber R. Zechner R. Kohlwein S.D. Obese yeast: triglyceride lipolysis is functionally conserved from mammals to yeast.J. Biol. Chem. 2006; 281: 491-500Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Among these lipases, Tgl2 is highly reactive toward both C8:0/C8:0/C8:0 TAG and C8:0/C8:0 DAG (16.Ham H.J. Rho H.J. Shin S.K. Yoon H-J. The TGL2 gene of Saccharomyces cerevisiae encodes an active acylglycerol lipase located in the mitochondria.J. Biol. Chem. 2010; 285: 3005-3013Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). We purified Tgl2 protein from the yeast whole cell lysates and performed a lipase assay using C8:0/C8:0/C8:0 TAG (glyceryl trioctanoate) as substrate. Before ESI-MS analysis, esterification of assay products was carried out by using C7 and C9, as described in the Materials and Methods. The amount of TAG, DAG, or MAG following the Tgl2 lipase assay was determined from the initial concentration of TAG (0.7 mM) after measuring the relative abundance of [TAG + Na]+ at m/z 494.2, [TAG′ + Na]+ at m/z 480.1/508.1, or [TAG″ + Na]+ at m/z 466.1/522.2 in the mass spectra (Fig. 4). Reaction yields for DAG-to-TAG′ and MAG-to-TAG″ esterifications using C7 and C9 were taken into account in the conversion of the relative abundances of TAG′ and TAG″ to the concentrations of DAG and MAG, respectively. The TAG′ and TAG″ production yields of 100% and 97% in the case of C7 (Fig. 1A) or those of 84% and 70% in the case of C9 (Fig. 1B), respectively were reflected in the calculated concentrations of DAG and MAG (Table 5).TABLE 5Concentration of the Tgl2 assay products measured by ESI-MS after esterification using C7 and C9Lipid SpeciesC7C9[TAG]aReaction yields for the TAG′ and TAG″ productions using C7 and C9 are taken into account.0.44 ± 0.020.46 ± 0.01[DAG] = [TAG′]aReaction yields for the TAG′ and TAG″ productions using C7 and C9 are taken into account.0.07 ± 0.010.09 ± 0.02[MAG] = [TAG″]aReaction yields for the TAG′ and TAG″ productions using C7 and C9 are taken into account.0.19 ± 0.010.15 ± 0.02[FA]from TAG = [DAG] × 1 + [MAG] × 20.45 ± 0.020.39 ± 0.04[FA]from DAG = [MAG] × 10.19 ± 0.010.15 ± 0.02Initial concentration of TAG at the start of lipase assay was 0.7 mM. Also listed are the concentrations of FA liberated from TAG and DAG. Values are shown in millimoles and include instrumental error (5%).a Reaction yields for the TAG′ and TAG″ productions using C7 and C9 are taken into account. Open table in a new tab Initial concentration of TAG at the start of lipase assay was 0.7 mM. Also listed are the concentrations of FA liberated from TAG and DAG. Values are shown in millimoles and include instrumental error (5%). Next, we calculated the amounts of FAs liberated from TAG and DAG to measure specific activities of Tgl2 lipase toward TAG and DAG, respectively. Because the stoichiometric amount of FA derived from TAG is the sum of [DAG] × 1 and [MAG] × 2, as listed in Table 5, the specific activity of Tgl2 toward TAG can be obtained by using the equation {([TAG]0 – [TAG]) × volume (380 μl)/[enzyme amount (4 μg) × reaction time t (60 min)] × [FA]from TAG/([DAG] + [MAG])}. The calculated specific activity toward TAG was 0.71 ± 0.03 μmol min−1 mg−1 using C7 and 0.62 ± 0.03 μmol min−1 mg−1 using C9 (Table 6).TABLE 6Specific enzyme activity of the yeast Tgl2 lipaseLipidsSpecific Lipase Activity (μmol min−1 mg−1)C7C9TAG0.71 ± 0.030.62 ± 0.03DAG1.06 ± 0.050.99 ± 0.06 Open table in a new tab To calculate the specific lipase activity toward DAG, we need to know the amount of DAG consumed by lipolysis when the initial concentration of DAG was 0.7 mM. To this end, we examined the reaction kinetics of Tgl2 lipolysis. The lipolysis of Tgl2 can be considered as consecutive first-order reactions: TAG→k1DAG→k2MAG where k1 and k2 are the rate constants. Because we know the initial concentration of TAG and the final concentrations of TAG, DAG, and MAG after 60 min lipolysis (Table 5), we can calculate the rate constants k1 and k2 from the following equations for consecutive reactions (20.Houston P.L. McGraw-Hill, New York, NY2001Google Scholar): [TAG]=[TAG]0exp(−k1t)[DAG]=[TAG]0k1k2−k1[exp(−k1t)−exp(−k2t)][MAG]=[TAG]0−[TAG]−[DAG] Rate constants k1 and k2 using C7 were 0.46 and 3.18 h−1, respectively, and k1 and k2 using C9 were 0.42 and 2.46 h−1, respectively. Thus, the Tgl2 lipase carried out the DAG hydrolysis six to seven times faster than the hydrolysis of TAG. Because the amount of DAG consumed by lipolysis, [DAG]0 – [DAG], is equal to [DAG]0 × {1 − exp [−k2 × t (60 min)]} and the stoichiometric amount of FA derived from DAG is [MAG] × 1, the specific activity of Tgl2 toward DAG can be calculated by using the equation {([DAG]0 − [DAG]) × volume (380 μl)/[enzyme amount (4 μg) × reaction time t (60 min)] × [FA]from DAG/[MAG])}. The specific activity toward DAG was 1.06 ± 0.05 μmol min−1 mg−1 using C7 and 0.99 ± 0.06 μmol min−1 mg−1 using C9 (Table 6). The specific lipase activity of Tgl2 toward DAG was 1.5–1.6 times higher than that toward TAG, showing a good agreement with the previous result (16.Ham H.J. Rho H.J. Shin S.K. Yoon H-J. The TGL2 gene of Saccharomyces cerevisiae encodes an active acylglycerol lipase located in the mitochondria.J. Biol. Chem. 2010; 285: 3005-3013Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Therefore, we have successfully measured both TAG and DAG hydrolyzing activities of the yeast Tgl2 lipase and demonstrated the feasibility of this method for biological samples. In conclusion, we describe a method enabling simultaneous quantification of TAG, DAG, and MAG after lipase assay without the aid of radioactive or fluorescent labeling. This method is based on a quantification strategy combining chemical esterification and ESI-MS and provides quantitative data for all products of lipase assay, including FA. Resulting information will allow the determination of lipolytic activities in the biological systems. Download .pdf (.08 MB) Help with pdf files 1,2-dioctanoyl-sn-glycerol 1-octanoyl-rac-glycerol glyceryl trioctanoate diacylglycerol dichloromethane hemagglutinin monoacylglycerol N-bromosuccinimide triacylglycerol